This invention relates to porous, hydrogel coatings useful for the immobilization of biologically active molecules and, particularly, to crosslinked, azlactone-functional hydrogel coatings that are useful for the preparation of DNA and protein arrays, diagnostic devices and materials for the separation of biological species. More particularly, this invention relates to porous hydrophilic, crosslinked, azlactone-functional coatings and gels.
Coatings derived from copolymers of polymerizable azlactones and olefinically unsaturated monomers are known. Such coatings are derived, in general, from rigid, high glass transition temperature (Tg), hydrophobic copolymers. Crosslinking is accomplished by dispersing or dissolving the azlactone copolymer with a crosslinking agent, generally in an approximately stoichiometric amount to the azlactone, in a volatile organic liquid, applying the mixture to a substrate, then allowing the coating to crosslink via azlactone ring-opening reactions with the crosslinking agent. Suitable crosslinkers are polyols and polyamines. Polyamines, such as ethylene diamine, react with azlactones at room temperature, thereby forming crosslinks. Because of the rapid reaction between azlactones and primary amines, incorporation of a ketone solvent in the coating mixture is desirable. Polyols react much slower with azlactones and generally require a catalyst, such as a strongly acidic or basic catalyst, to promote crosslinking.
Various coatings derived from azlactone copolymers are known. For example, known coatings include copolymers of 2-alkenylazlactones with acrylic acid esters and copolymerizable vinylidene compounds having at least one hydroxyl group that crosslink on drying or mild heating. Such polymers crosslink by reaction of the hydroxyl groups on one chain of the polymer with azlactone groups on other chains. In general, an acidic or basic catalyst is again needed to facilitate the crosslinking reaction. Coatings derived from azlactone copolymers that are crosslinkable by exposure to radiation are useful in imaging applications. Uncrosslinked azlactone copolymers may be used to coat a variety of substrates. These coated substrates can be used for the immobilization of functional materials, including biologically active species such as proteins. Crosslinked azlactone-functional moieties may be included in a coating over the surfaces of chemically reactive, porous supports. These reactive supports can, in turn, be reacted with biologically active materials to produce adduct supports.
While there are a variety of methods for producing coatings derived from azlactone-functional materials, some of which provide azlactone-functional coatings useful for the immobilization of other species, there remains a need for additional or improved methods for providing coated materials for use in the immobilization of biologically active materials.
This invention relates to the preparation of reactive hydrophilic coatings and hydrogels that can be applied to various substrates for the purpose of covalently attaching a functional material to the substrate. In particular, the invention provides a crosslinked hydrogel for coating a substrate comprising at least one azlactone-functional copolymer comprising a plurality of azlactone moieties, a plurality of azlactone functional groups, and at least one comonomer, and at least one crosslinker comprising a first moiety and a second moiety, wherein the first moiety of a first crosslinker is covalently bound to a first azlactone moiety and the second moiety of the first crosslinker is covalently bound to a second azlactone moiety or a second crosslinker.
In some embodiments of the crosslinked hydrogel of the present invention, the second moiety of the first crosslinker is covalently bound to a second azlactone moiety. In such embodiments, the first crosslinker may be a primary polyamine, a polyether polyamine, a compound containing both a primary and a secondary amine, or any other suitable crosslinker. In other embodiments, the second moiety of the first crosslinker is covalently bound to a second crosslinker molecule. In such embodiments, the first crosslinker may be bound to a second crosslinker molecule having the same chemical structure as the first crosslinker. Alternatively, the first crosslinker may be bound to a second crosslinker having a different chemical structure than the first crosslinker. In either embodiment described above, the first crosslinker, the second crosslinker, or both may be a heterobifunctional crosslinker such as an aminoalkylalkoxysilane.
In some embodiments of the present invention, the crosslinked hydrogel includes polymers made from ionic or non-hydrophilic comonomers.
This invention relates to the preparation of reactive hydrophilic coatings and hydrogels that can be placed on the surfaces of various substrates or within the structures of various structured (i.e., macro- or microstructured) substrates for the purpose of covalently attaching a functional material to the substrate. More specifically, the present invention provides compositions and processes for applying coatings including azlactone functionality onto substrate surfaces. The coatings may include thin films, thick gels, or any intermediate thickness. These coatings may provide for the attachment of functional materials to the substrate. A xe2x80x9cfunctional materialxe2x80x9d is any chemical species having (a) a nucleophilic group that can react with an azlactone and (b) another reactive site, which is desired to be attached to the substrate to accomplish a specific purpose. In certain embodiments of the present invention, the functional material includes a biologically active material.
For the purposes of this invention, the following definitions shall have the meanings set forth.
xe2x80x9c1xc2x0/2xc2x0 amine-containing compoundxe2x80x9d as used herein shall mean any compound, molecule, composition or complex having one primary amine-containing functional group and at least one secondary amine-containing functional group.
xe2x80x9cAzlactone functional groupxe2x80x9d shall mean a functional group having the structure: 
wherein R1 and R2 are, independently, an alkyl group having 1-14 carbon atoms, a cycloalkyl group having 3-14 carbon atoms, an aryl group having 5-12 ring atoms, an arenyl group having 6-26 carbon atoms and 0-3 S, N, or nonperoxidic O atoms, or R1 and R2 taken together with the carbon to which they are both joined form a carbocyclic ring having 4-12 carbons, and n is the integer 0 or 1.
xe2x80x9cFunctional groupxe2x80x9d as used herein shall mean a combination of atoms in a molecule, compound, composition or complex that tends to function as a single chemical entity. Examples of functional groups include, but are not limited to, xe2x80x94NH2 (amine), xe2x80x94COOH (carboxyl), siloxane, xe2x80x94OH (hydroxyl), and azlactone. For example, prior to reaction, certain crosslinkers may contain one or more amine functional groups and certain copolymers may contain one or more azlactone functional groups.
xe2x80x9cHeterobifunctionalxe2x80x9d as used herein shall mean, with respect to any molecule, compound, composition or complex, having more than one functional group and having at least two functional groups that are different from one another. For example, an amino acid is heterobifunctional because it contains two functional groups, the amino group and the carboxyl group, that are different than one another.
xe2x80x9cHydrogelxe2x80x9d means a water-containing gel, i.e., a polymer that is hydrophilic and will absorb water, yet is insoluble in water.
xe2x80x9cIonic,xe2x80x9d with respect to monomers, shall be construed broadly to refer to monomers that inherently have a formal charge as well as monomers that are acidic or basic enough that they can acquire a formal charge when in contact with an aqueous medium.
xe2x80x9cMoietyxe2x80x9d as used herein shall mean the portion of a functional group from a first reactant that combines with a functional group of a second reactant to form a covalent bond in the reaction product. For example, in a peptide bond, the xe2x80x94NHxe2x80x94 that participates in the peptide bond remains from the amine functional group of one amino acid and is therefore considered, herein, to be an amine moiety in the peptide product. The xe2x80x94Cxe2x95x90O that participates in the peptide bond remains from the carboxylic acid functional group of the second amino acid and is therefore considered a carboxyl moiety in the peptide product.
xe2x80x9cNon-hydrophilicxe2x80x9d as used herein shall refer, with respect to any molecule, compound, composition or complex, to any material that has a Hydrophilicty Index of less than about 40.
xe2x80x9cPot lifexe2x80x9d shall mean the length of time during which a coating formulation remains soluble and homogeneous with low attendant viscosity.
xe2x80x9cPrimerxe2x80x9d shall mean any suitable material that promotes or improves adhesion between the copolymer and the substrate. xe2x80x9cPrimerxe2x80x9d shall include both inert primers and reactive primers. Inert primers act as an adhesion-promoting interlayer between the copolymer and the substrate. Reactive primers form covalent bonds between the copolymer and the substrate to improve adhesion.
The azlactone-functional hydrogel coatings of the present invention are produced by first preparing a solution of a hydrophilic, azlactone-functional copolymer. This copolymer is then formulated with an appropriate crosslinker, and the mixture is then coated on or applied to an appropriate substrate. The crosslinker reacts with a portion of the azlactone groups of the copolymer, thereby forming the porous, crosslinked hydrogel. Unreacted azlactone groups in the hydrogel coating are then available for the attachment of functional materials for the appropriate end uses.
Azlactone-functional copolymers may be prepared by a variety of free radical polymerization processes in which alkenyl azlactone monomers are copolymerized with comonomers. Typical solution polymerization processes have been reported, for example, in U.S. Pat. No. 4,304,705, issued to Heilmann et al. and U.S. Pat. No. 3,583,950, issued to Kollinsky et al. For the purposes of this invention, suitable comonomers include, without limitation, hydrophilic or water-soluble monomers such as acrylamide, methacrylamide, N-mono- and N,N-disubstituted acrylamides and methacrylamides, N-vinylamides such as N-vinylformamide and N-vinylpyrrolidinone, and hydroxyalkylacrylates and acrylamides such as 2-hydroxyethylmethacrylate and N-acryloyl-trishydroxymethylaminomethane. For many applications, uncharged copolymers may be desirable in order to reduce the possibility for nonspecific binding of biological macromolecules to the coatings. For specific applications, however, ionic comonomers may also be incorporated into the copolymers. Ionic monomers may be anionic or cationic. Anionic monomers include unsaturated acids and their metal salts, such as acrylic, methacrylic, maleic, fumaric, and itaconic acids, vinyl phosphoric and phosphonic acids, styrenesulfonic acid, and 2-acrylamido-2-methyl-1-propanesulfonic acid; cationic monomers include amine-containing monomers such as 2-, 3- or 4-vinylpyridine, (3-acrylamidopropyl)trimethylammonium chloride, 2-diethylaminoethylacrylate and methacrylate, 3-dimethylarninopropylacrylate and methacrylate, and similarly substituted acrylamides and methacrylamides.
Copolymers within the scope of this invention also may include non-hydrophilic comonomers. As used herein, non-hydrophilic comonomers include any comonomer that has a Hydrophilicity Index of less than about 40. The Hydrophilicity Index (or xe2x80x9cH.I.xe2x80x9d) is an empirical concept that may be useful for describing the hydrophilic character of monomers suitable for use in the present invention. H.I. is defined as:       H    .    I    .    =                                                        total  molecular  weight  of  all  hydrophilic                                                                          groups  in  the  monomer                                                  molecular  weight  of  the  monomer              xc3x97    100.  
H.I. =total molecular weight of all hvdrophilic groups in the monomer x 100. molecular weight of the monomer
Hydrophilic groups are generally those that are functionally capable of forming hydrogen bonds with water. Examples of hydrophilic groups include, but are not limited to, xe2x80x94Nxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94NH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94OH, xe2x80x94COOH, xe2x80x94Cxe2x95x90O, xe2x80x94OCxe2x95x90O, xe2x80x94CO2xe2x88x92M+ (wherein M+ is an alkali or alkaline earth metal cation), xe2x80x94SH, SO3H, xe2x80x94SO3xe2x88x92M+, xe2x80x94NHCONHxe2x80x94, and other ionic functional groups.
Non-hydrophilic comonomers may be incorporated at less than about 50% by weight and still maintain sufficient hydrophilicity of the coatings and help minimize nonspecific binding. Certain embodiments incorporate non-hydrophilic comonomers at less than about 30% by weight. Suitable non-hydrophilic comonomers include, without limitation, known acrylate and methacrylate esters, styrene, and other free radically polymerizable monomers.
Once the appropriate azlactone-functional copolymer has been prepared, coating mixtures are formulated by adding crosslinkers to the copolymer. This is conveniently done in an appropriate organic solvent that is nonreactive with azlactone functional groups. The copolymer may be diluted with solvent to a concentration of about 5% by weight or less prior to the addition of crosslinker. In other embodiments, the copolymer may be diluted with solvent to concentrations of about 10% by weight or about 20% by weight prior to the addition of crosslinker. The solvent used for dilution may be the same solvent in which the copolymer was prepared or may be one or more different solvents. Crosslinking, or gellation time, is conveniently controlled by copolymer concentration and the amount of crosslinker added, thereby allowing adequate time for coating or for filling structures, followed by rapid cure time to provide finished product. In general, the lower the copolymer concentration or the lower the amount of crosslinker, the longer it will take for the crosslinking/gellation to occur.
Crosslinkers useful for the purposes of the present invention include, without limitation, materials that include nucleophilic groups that will undergo ring-opening reactions with azlactone functional groups. Suitable crosslinkers include primary polyamines, such as ethylenediamine, 1,3-propanediamine, 1,3-diamino-2-hydroxypropane, 1,6-hexanediamine, tris-(2-aminoethyl)amine, and the like; and polyetherpolyamines, such as 4,7,10-trioxa-1,13-tridecanediamine, 3,6-dioxa-1,8-diaminooctane, amine-terminated polyethyleneglycol and polypropyleneglycol homopolymers and copolymers, and the like. To achieve the purposes of the invention, the stoichiometry between the nucleophilic groups of the crosslinker and the azlactone functional groups of the copolymer should be less that 1:1 so that the final crosslinked hydrogel still contains reactive azlactone functionality. Thus, the azlactone content in the original copolymer will provide an upper limit on the amount of crosslinker that may be added to the coating formulation. The intended final use of the hydrogel may also dictate, to a certain degree, the amount of crosslinker used in the formulation. The amount of crosslinking will influence the swelling and porosity of the hydrogel, thus affecting the rate of diffusion of reagents or target molecules into and out of the hydrogel. Generally, less crosslinking provides a hydrogel having larger pores, thereby allowing diffusion of larger biological macromolecules through the hydrogel.
As indicated above, gellation time can be controlled to a certain extent by controlling the concentrations of copolymer and crosslinker. As used herein, gellation time refers to the amount of time necessary for a solution that can form a gel to become no longer fluid. In many instances, these parameters provide adequate control to allow placing the coating solution into the proper configuration prior to the occurrence of gellation. For some applications or product concepts, however, these parameters by themselves do not allow long enough gel times for use in manufacturing. Through the use of some novel crosslinking schemes, the present invention now provides coating formulations with extended pot lives; that is, the coating formulations remain soluble and homogeneous with low attendant viscosities for extended periods of time. Upon evaporation of the solvent and/or raising the temperature of the coated substrate, the coating formulations crosslink to produce the hydrogels of the present invention. These novel crosslinking schemes are achieved by using heterobifunctional crosslinkers, i.e., crosslinkers that have one nucleophilic functional group that reacts with the azlactone group at ambient temperature in solution (e.g., a primary amine) and at least one other functional group that can lead to a crosslinking reaction upon removal of the solvent or upon raising the temperature. One class of crosslinkers that may be used in this manner is the aminoalkylalkoxysilanes such as, for example, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N-[3-(trimethoxysilyl)propyl]ethylenediamine, or other aminoalkylmono-, di- and tri-alkoxysilanes. The amino group undergoes a ring-opening addition reaction with an azlactone group, providing a pendant alkoxysilane group on the copolymer. Upon dry-down, the alkoxy groups may be hydrolyzed and subsequently form siloxane crosslinks between polymer chains. Depending upon the nature of the substrate, covalent bonds may simultaneously be made with functional groups on the surface of the substrate (for example, if the substrate is siliceous, siloxane linkages to the substrate may be formed).
Another class of crosslinking agents useful for prolonging pot life or gellation times includes primary/secondary (1xc2x0/2xc2x0) amine-containing compounds. In these materials, the primary amine provides rapid reaction with an azlactone group on the copolymer at room temperature, while the secondary amine is relatively slow to react. Removing the solvent, raising the temperature of the coated article, or both allows the secondary amine to react to form the hydrogel. Suitable 1xc2x0/2xc2x0 amine-containing compounds include, without limitation, N-methyl-1,2-ethanediamine, N-ethyl-1,2-ethanediamine, N-isopropyl-1,2-ethanediamine, and other N-alkyldiaminoalkanes. Increasing the steric bulk of the N-alkyl substituent provides a greater barrier to reaction of the secondary amino group, thus necessitating a higher temperature to produce crosslinking.
Once formulated, the coating solutions can be applied to desired substrates and dried (optionally with the application of heat) to produce the hydrogels of the present invention. Coating methods can vary widely depending upon the particular substrate, and may be selected from methods known in the art. These include, for example, extrusion coating, die coating, dip coating, air-knife coating, gravure coating, curtain coating, spray coating, use of wire-wound coating rods, and the like.
With certain substrates, the hydrophilic azlactone-functional polymer will exhibit fairly good adhesion. Crosslinking to produce a hydrogel insolubilizes the coating and reduces the likelihood of the coating coming off of the substrate in subsequent manipulations. Adhesion of the coating to the substrate may be improved, if desired, by any known method. Such methods include, but are not limited to, various pre-treatments to or coatings on the surface of the substrate, such as corona or plasma treatments, or by the application of primers. Suitable primers include, without limitation, polyethylenimine, polyvinylidenechloride, primers such as those reported in U.S. Pat. No. 5,602,202, issued to Groves, and colloidal dispersions of inorganic metal oxides in combination with ambifunctional silanes such as those reported in U.S. Pat. No. 5,204,219, issued to Van Ooij et al., U.S. Pat. No. 5,464,900, issued to Stofko et al., and U.S. Pat. No. 5,639,546, issued to Bilkadi. Other methods of increasing adhesion to polyolefin substrates are reported in U.S. Pat. No. 5,500,251, issued to Burgoyne et al.
The hydrophilic coatings and hydrogels of the invention may be applied to a wide variety of substrates. The substrates may be natural or synthetic, organic or inorganic, porous or nonporous, flat and substantially featureless or highly structured. The substrates may be film-like, particulate-like, or molded plastic articles. Suitable substrates include, without limitation, standard 96-, 384-, or 1536-well plastic microtiter plates, including filtration plates; grooved, microreplicated films; microfluidic channels in microfluidic devices; embossed or microstructured films; tubes or capillaries; spin tubes or spin columns; glass, ceramic, or metal particles or fibers, including porous particles or fibers; porous or nonporous polymeric fibers or particles, such as chromatographic particles; oriented or non-oriented polymeric films; woven or nonwoven webs (such as fibrous webs); porous or microporous membranes; and the like.
The substrate chosen will depend upon the intended application or device. Those applications include, without limitation, devices such as DNA or protein arrays; biological assay or diagnostic devices; capillary electrophoresis, electrochromatography, or other separation devices; chromatographic supports for affinity, ion exchange, hydrophobic interaction, or other types of separations and purifications; cell selection or separation devices; and the like. For example, when using oriented polymeric films as the substrate, the coatings of the invention are advantageously used to prepare high-density, miniaturized arrays as described in International Publication Number WO 99/53319.
Once the reactive coatings or hydrogels are applied to the substrates, the residual azlactone functionality is available for reaction with the functional material. Again, the intended application will dictate the identity of the functional material. Preferred functional materials are biologically active materials such as proteins, enzymes, oligonucleotides, or any other species that may interact with biological species. Derivatization may be conducted in aqueous, buffered media, as is well known for reactions of azlactone-functional substrates, although other media such as organic solvents may be used.